TY - JOUR
T1 - Identification of nine cryptic species of Candida albicans, C. glabrata, and C. parapsilosis complexes using one-step multiplex PCR
AU - Arastehfar, Amir
AU - Fang, Wenjie
AU - Pan, Weihua
AU - Liao, Wanqing
AU - Yan, Liang
AU - Boekhout, Teun
PY - 2018/9/25
Y1 - 2018/9/25
N2 - BACKGROUND: Candida albicans, Candida glabrata, and Candida parapsilosis are three prevalent causes of candidiasis, worldwide. These species are considered as nine medically important complex species. Limited knowledge about these newly recognized species prompted us to develop a one-step, multiplex PCR to detect and identify them in clinical settings.METHODS: Primers targeting Hyphal Wall Protein I gene for the C. albicans, C. dubliniensis, C. africana, Intergenic Spacer for the C. glabrata, C. nivariensis, C. bracarensis, and Intein and ITS rDNA for the C. parapsilosis, C. orthopsilosis, and C. metapsilosis were designed. Using 168 CBS reference strains and 280 clinical isolates, the specificity and reproducibility of the developed assay were evaluated.RESULTS: Our developed assay successfully identified and distinguished all the nine species. No cross-reaction with closely- and distantly-related yeast species, Aspergillus species and human DNA was observed, resulting in 100% specificity. The ambiguous results obtained by MALDI-TOF for C. albicans and C. africana were corrected by our 9-plex PCR assay. This assay identified all the cryptic complex species from two test sets from Iran and China, correctly.CONCLUSIONS: Our developed multiplex assay is accurate, specific, cost/time-saving, and works without the tedious DNA extraction steps. It could be integrated into routine clinical laboratories and as a reliable identification tool and has the potential to be implemented into epidemiological studies to broaden the limited knowledge of cryptic species complexes.
AB - BACKGROUND: Candida albicans, Candida glabrata, and Candida parapsilosis are three prevalent causes of candidiasis, worldwide. These species are considered as nine medically important complex species. Limited knowledge about these newly recognized species prompted us to develop a one-step, multiplex PCR to detect and identify them in clinical settings.METHODS: Primers targeting Hyphal Wall Protein I gene for the C. albicans, C. dubliniensis, C. africana, Intergenic Spacer for the C. glabrata, C. nivariensis, C. bracarensis, and Intein and ITS rDNA for the C. parapsilosis, C. orthopsilosis, and C. metapsilosis were designed. Using 168 CBS reference strains and 280 clinical isolates, the specificity and reproducibility of the developed assay were evaluated.RESULTS: Our developed assay successfully identified and distinguished all the nine species. No cross-reaction with closely- and distantly-related yeast species, Aspergillus species and human DNA was observed, resulting in 100% specificity. The ambiguous results obtained by MALDI-TOF for C. albicans and C. africana were corrected by our 9-plex PCR assay. This assay identified all the cryptic complex species from two test sets from Iran and China, correctly.CONCLUSIONS: Our developed multiplex assay is accurate, specific, cost/time-saving, and works without the tedious DNA extraction steps. It could be integrated into routine clinical laboratories and as a reliable identification tool and has the potential to be implemented into epidemiological studies to broaden the limited knowledge of cryptic species complexes.
KW - Candida albicans/genetics
KW - Candida glabrata/genetics
KW - Candida parapsilosis/genetics
KW - Candidiasis/diagnosis
KW - DNA, Fungal/isolation & purification
KW - DNA, Ribosomal/analysis
KW - Humans
KW - Multiplex Polymerase Chain Reaction
KW - Reproducibility of Results
KW - Sequence Analysis, DNA
KW - Species Specificity
KW - Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
U2 - 10.1186/s12879-018-3381-5
DO - 10.1186/s12879-018-3381-5
M3 - Article
C2 - 30253748
SN - 1471-2334
VL - 18
SP - 480
JO - BMC Infectious Diseases
JF - BMC Infectious Diseases
IS - 1
ER -